RESUMO
We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Cisteína Endopeptidases/metabolismo , Antígenos HLA-A/metabolismo , Complexos Multienzimáticos/metabolismo , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Sequência de Bases , Linhagem Celular Transformada , Citotoxicidade Imunológica , Primers do DNA/genética , Epitopos/genética , Epitopos/metabolismo , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Células Tumorais CultivadasRESUMO
To use peptides as synthetic vaccines, they have to be coupled to a carrier protein to make them more immunogenic. Coupling efficiency between a carrier protein and a peptide, however, is difficult to control with respect to loading density of the peptide. This makes these carrier proteins poorly suitable for practicle use. Attempts have been reported to find carrier molecules or delivery systems which allow easy coupling or incorporation of peptides, reproducible loading densities and well defined products. We compared several promising constructs or delivery systems by immunization of male pigs using a tandem GnRH peptide as a branched polylysine construct, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipoamide peptide in an immuno-stimulating complex (ISCOM). We found the lipo-thioester and the branched polylysine constructs to be the most effective carrier molecules for the induction of antibodies against GnRH and immunocastration of pigs.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Hormônio Liberador de Gonadotropina/imunologia , Peptídeos/administração & dosagem , Vacinas Sintéticas/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Relação Dose-Resposta Imunológica , Emulsões , Adjuvante de Freund/administração & dosagem , Hemocianinas/administração & dosagem , Masculino , Orquiectomia , Tamanho do Órgão , Ácido Palmítico/administração & dosagem , Ácido Palmítico/metabolismo , Peptídeos/genética , Peptídeos/imunologia , Polilisina/administração & dosagem , Radioimunoensaio , Sequências Repetitivas de Aminoácidos/imunologia , Suínos , Testículo/imunologia , Ácido Tióctico/administração & dosagem , Ácido Tióctico/análogos & derivados , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genéticaRESUMO
The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester-, a disulfide-, an amide- and a thioether bond were investigated. As carrier molecules a peptide, an N-palmitoylated peptide or a C(16)-hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide >> thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide-based vaccine than a bond that is less susceptible to biological degradation.
Assuntos
Anticorpos/imunologia , Portadores de Fármacos/química , Hormônio Liberador de Gonadotropina/imunologia , Hidrocarbonetos/imunologia , Parvovirus Canino/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Amidas/química , Animais , Anticorpos/química , Dissulfetos/química , Feminino , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/imunologia , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/genética , Cobaias , Hidrocarbonetos/química , Masculino , Modelos Químicos , Ácido Palmítico/química , Parvovirus Canino/química , Peptídeos/química , Peptídeos/imunologia , Sulfetos/química , SuínosRESUMO
Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.
Assuntos
Hormônio Foliculoestimulante , Oxirredutases , Receptores do FSH/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Gonadotropina Coriônica/química , AMP Cíclico/biossíntese , Desenho de Fármacos , Hormônio Foliculoestimulante/análogos & derivados , Hormônio Foliculoestimulante/antagonistas & inibidores , Hormônio Foliculoestimulante/metabolismo , Glutarredoxinas , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Conformação Proteica , Proteínas/química , Ratos , Receptores do FSH/metabolismo , Células de Sertoli/metabolismo , Relação Estrutura-Atividade , Tiorredoxinas/químicaRESUMO
Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBut) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.
Assuntos
Antígenos/imunologia , Ácido Palmítico/metabolismo , Peptídeos/imunologia , Compostos de Sulfidrila/metabolismo , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Ésteres , Cobaias , Imunização , Masculino , Dados de Sequência Molecular , Peptídeos/síntese química , SuínosRESUMO
Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.
Assuntos
Proteínas do Capsídeo , Vírus da Panleucopenia Felina/imunologia , Vison/virologia , Infecções por Parvoviridae/prevenção & controle , Infecções por Parvoviridae/veterinária , Parvovirus Canino/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Capsídeo/genética , Capsídeo/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/imunologia , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Virais/administração & dosagemRESUMO
Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.
Assuntos
Adenovírus Humanos/imunologia , Apresentação de Antígeno , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/imunologia , Adenovírus Humanos/genética , Adjuvantes Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Linhagem Celular , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/fisiologia , Relação Dose-Resposta Imunológica , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Indução Enzimática/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/fisiologia , Biossíntese Peptídica/imunologia , Complexo de Endopeptidases do Proteassoma , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia , Tetraciclina/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
CTL directed against the Moloney murine leukemia virus (MuLV) epitope SSWDFITV recognize Moloney MuLV-induced tumor cells, but do not recognize cells transformed by the closely related Friend MuLV. The potential Friend MuLV epitope has strong sequence homology with Moloney MuLV and only differs in one amino acid within the CTL epitope and one amino acid just outside the epitope. We now show that failure to recognize Friend MuLV-transformed tumor cells is based on a defect in proteasome-mediated processing of the Friend epitope which is due to a single amino acid substitution (N-->D) immediately flanking the C-terminal anchor residue of the epitope. Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules. Thus, a potential CTL epitope present in the Friend virus sequence is not properly processed and presented because of a natural flanking aspartic acid that obliterates the correct C-terminal cleavage site. This constitutes a novel way to subvert proteasome-mediated generation of proper antigenic peptide fragments.
Assuntos
Substituição de Aminoácidos/imunologia , Cisteína Endopeptidases/metabolismo , Epitopos de Linfócito T/metabolismo , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Vírus da Leucemia Murina de Friend/imunologia , Antígenos H-2/metabolismo , Células HeLa , Humanos , Hidrólise , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/imunologia , Fragmentos de Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Vírus Rauscher/imunologia , Linfócitos T Citotóxicos/enzimologia , Células Tumorais CultivadasRESUMO
Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.